Nanoflow liquid chromatography (“nanoLC”) is a technique for resolving very complex samples that are limited in concentration or volume. Predominantly the technique is used for proteomic studies where it is often used in combination with mass spectrometry. NanoLC of either whole proteins or a proteolytic digest is performed to separate very complex samples, and then the flow from the nanoLC is directed into a mass spectrometer. The advantages of nanoLC mass spectrometry as compared to conventional higher flow rate chromatography include lower sample volume requirements and higher sensitivity. NanoLC separation reduces the complexity of the sample by resolving the different components of a sample, allowing the mass spectrometer to obtain mass spectra for many components contained in the sample.
A liquid chromatography/mass spectrometry (LC/MS) system is composed of a pumping system, an autosampler injection system, a chromatography column(s), a means of ionization, and a mass spectrometer detector. The pumping system delivers mobile phase (solvents) at a user defined flow rate, typically ranging from 20 nL/min to 6 mL/min. Mobile phases generally consist of two solvents where one is predominantly aqueous (water) in nature and the other is predominantly organic in nature (methanol, acetonitrile, isopropanol, etc). Additives such as formic acid, acetic acid, ammonium acetate, ammonium hydroxide, etc, can also be present in the mobile phase. The pumping system delivers the different solvents of the mobile phase in a precise way that is referred to as the gradient. For an analysis using a reversed-phase column, the gradient typically begins with high aqueous solvent and gradually organic solvent is introduced, while the overall flow rate remains constant. By the end of the analysis, the mobile phase content is predominantly organic solvent.
The target sample of interest is introduced to the flow stream by the autosampler injection system. This system typically consists of an aspiration needle and a valve. The system aspirates the sample of interest using the needle and subsequently injects the sample into the injection valve. This valve has a sample loop that can be 1 microliter to 10 milliliters in volume, and is filled with the sample of interest. Then the valve is switched from the “load” position to the “inject” position, and the plug of sample from the loop becomes in-line with the flowing mobile phase from the pumping system. The mobile phase displaces the sample plug from the loop and pushes it into the analysis path. At this point the pumps would be early in the gradient program, so that the sample plug is in predominantly aqueous solvent.
The sample continues flowing downstream and reaches the column. As the mobile phase is predominantly aqueous in nature, the sample adsorbs to the stationary phase at the top of the reversed-phase column. As the analysis is performed the solvent composition of the mobile phase becomes increasingly organic in content. As this occurs, mass transfer of the retained molecules occurs between the stationary and mobile phases. The components of the sample make their way through the column at different rates, and thus the sample is chromatographically resolved.
The various components of the sample exit the column at different time points in the flowing mobile phase. As the mass spectrometer is only able to detect ions, not neutral molecules, the sample components must be converted to ions prior to entering the mass spectrometer. One means of generating ions is electrospray ionization, which at lower flow rates is referred to as nanoelectrospray ionization. In brief, a high voltage is applied to the column effluent containing the sample components of interest. The high voltage generates highly charged droplets and through subsequent droplet evaporation and droplet fission, desolvated ions are formed.
The ions then enter the mass spectrometer detector. The mass spectrometer determines the mass-to-charge ratio of the ions. Many of these instruments perform tandem mass spectrometric measurements, allowing structural information of the ion to be determined.
At the end of the analysis, the column is washed with high organic solvent and is then re-equilibrated in the aqueous mobile phase. An autosampler routine is used to wash the injection needle and sample loop several times to help minimize sample-to-sample cross-contamination and carryover. At this point a second analysis can be performed.
NanoLC is typically performed at flow rates between 5-500 nL/min. These low flow rates necessitate the use of special pumping systems, chromatography columns, and spray emitters used for the electrospray ionization (“ESI”) interface to the mass spectrometer. Even with the specialty equipment currently available nanoLC is very difficult to perform. Making connections with micron size tubing requires user intervention to tighten fittings. As there is no feedback in the system, the user must guess the correct amount of tightening to make the fitting leak-free. Generally, this leads to over tightening of the fittings which may prevent leaking, however, simultaneously creates a secondary problem. The over tightening can either damage the tube, the fitting, or the fragile capillaries. Connecting nanoLC columns is especially difficult as the fragile columns are prone to damage due to frits, stationary phase within the column, or other material in the tube being crushed, cracked, or over compressed. Additionally, the rotation of the conventional fitting can cause the tube or capillary to be twisted, resulting in grinding or damage from the twisting itself.
Connections between the pumping system and column, and between the column and spray emitter are especially prone to leaks. Often the leaks are very difficult to detect as evaporation renders the leak unperceivable at the low flow rate or the liquid build up is so small it is difficult to observe. This holds true for both chip-based microfluidics and conventionally assembled components. Leaks at these connections can be due to user error in making connections, or due to a change in the system backpressure which can result from a clogged column or spray emitter. Further challenges of nanoLC include column irreproducibility, spray irreproducibility, poorly optimized solvent gradient separation, insufficient column regeneration period, and poorly optimized emitter position. Conventional fittings are thread-based requiring rotation of the ferrule, and subsequently the tubing, to generate a seal. The applied twisting motion causes tubing ends to grind against surfaces creating jagged ends and producing particulates that subsequently clog and contaminate fragile components downstream. The challenges associated with nanoLC results in the technique only being successfully used by very few expert users.
Although various research and apparatuses have attempted to reduce the difficulty of conducting nanoLC there is still a need for a simple, robust system with easy to change components and integrated diagnostic sensors for identifying malfunctions in the dynamic fluidic system. This includes conventional, microfluidic, and nanofluidic-based fluidic systems.
Additionally, current technologies provide limited measurements at the pumping level, which is not indicative of component status at the chromatography and electrospray level. Therefore, the current technology lacks the ability to indicate the malfunction location. In addition to not being able to diagnose the problem, these current technologies lack the ability to automatically change-out the appropriate components due to both lack of information and due to instrument design involving connecting fittings that require human intervention.
Therefore there exists a need for an automatic sealing device for making connections in fluidic systems to reduce potential user over tightening and to self-align the components within the fluidic system.
There also exists a need for a prefabricated insert containing multiple fluidic components to reduce the number of connects that a user is required to make.
There exists a need for detecting leaks in microfluidic and nanofluidic applications where sample size and flow rates are too small to be detected by conventional means.
There further exists a need for fluidic system components that are easy to replace and can be interchanged by an automated process.